Cells of the renal medulla are exposed to extraordinarily high concentrations of the potent denaturant, urea, as a consequence of the renal concentrating mechanism. The molecular mechanisms through which renal medullary cells adapt to and tolerate this harsh environment have important implications for the understanding of water and urea physiology, and for the understanding of and potential enhancement of cellular protection from metabolic stress in diverse pathophysiological contexts. The applicant has obtained abundant evidence that, in marked contrast to the other principal medullary solute, NaC1, urea activates a novel receptor tyrosine kinase pathway in renal epithelial cells. Two candidate urea sensing molecules exhibiting prompt, solute- and tissue-specific, urea-inducible autophosphorylation or tyrosine phosphorylation have been partially purified by the applicant through co-immunoprecipitation with urea-responsive signaling molecules, and through lectin affinity precipitation, respectively. The overall objective of the project is to understand the molecular mechanism and physiological consequences of the activation of urea-inducible kinase signaling pathways in renal medullary cells in vitro and in vivo, and to identify these and other potentially novel urea sensing and effector molecules. The ability of known SH2 domain-containing receptor tyrosine kinase effector molecules to mediate elements of the renal epithelial cell adaptive response to urea stress (Aim 1) will be determined through inhibition of effector pathways with pharmacological agents and through transient and stable transfection with inducible dominant-negative expression constructs. The two candidate urea sensors will be partially biochemically purified (and other potential candidates identified, if necessary) using lysates prepared from 32Pi metabolically labeled control- and urea-treated cells, through chromatographic and batch affinity strategies in conjunction with 2-D gel electrophoresis (Aim II). Lastly, the candidate urea sensing proteins will be identified through tandem mass spectrometry, or, if novel, cloned through peptide microsequencing followed by cDNA library screening (Aim III). Thereafter, their physiological regulation at the transcriptional, translational, and post-translational levels will be determined in renal medullary cells in culture and in rodent models associated with abnormal water and urea metabolism.